Project Summary/Abstract Metalloproteins containing manganese in a redox-active role are involved in a variety of physiologically important reactions of dioxygen metabolism. These include, amongst others, a superoxide dismutase that detoxifies superoxide radicals to O2 and peroxide, a catalase that disproportionates peroxide to O2 and H2O, and perhaps the most complex and important, the Mn-containing oxygen-evolving complex (Mn-OEC) that is involved in the oxidation of water to dioxygen in photosystem II. The Mn-OEC generates almost all of the dioxygen that supports aerobic life, and it is abundant in the atmosphere because of its constant regeneration by the oxidation of water. The light-induced oxidation of water to dioxygen is one of the most important chemical processes occurring on such a large scale in the biosphere. The water-oxidation reaction involves removal of four electrons, in a stepwise manner by light-induced oxidation, from two water molecules by the Mn-OEC to produce a molecule of oxygen. Central questions that need to be resolved and the overall objective of this proposal are as follows: 1) Characterize the structure and the changes of the oxo-bridged heteronuclear Mn4Ca complex as it advances through the enzymatic cycle, 2) Elucidate the structural and functional role of the cofactors Cl- and Ca2+, 3) Determine the oxidation states and electronic structure of the Mn complex in the four intermediate S-states, 4) Determine the mechanism of water oxidation and oxygen evolution. The interplay between X-ray spectroscopy and EPR has played an essential role in our understanding of the structural and mechanistic aspects of O2 evolution. The samples for X-ray spectroscopy will be characterized by EPR. The structural changes of the Mn complex as it advances through the enzymatic cycle are determined by high-resolution XAS methods using samples prepared by flash illumination, and single- crystals of PS II. The oxidation states and electronic structure of the Mn complex are determined by Mn K- and L-edge, K[unreadable] emission, and X-ray resonant Raman spectroscopies.